Disrupted membrane lipid homeostasis is implicated in epithelial barrier dysfunction and inflammatory signaling in ulcerative colitis. Associated lipid alterations include a reduced phosphatidylcholine-to-phosphatidylethanolamine (PC/PE) ratio and accumulation of lysophosphatidylcholine (LPC) and ceramide. Although oleic acid (OA) has shown protective effects in experimental colitis, its therapeutic utility is limited by rapid metabolic turnover. We therefore tested whether 2-hydroxyoleic acid (2-OHOA), a synthetic hydroxylated OA derivative with enhanced metabolic stability, protects against intestinal inflammatory injury. In IFN-γ/TNF-α-stimulated Caco-2 monolayers, 2-OHOA pretreatment preserved epithelial barrier function, attenuated the increase in membrane fluidity, and reduced STAT3 and NF-κB activation, whereas OA at the same concentration provided little protection. In a dextran sulfate sodium (DSS)-induced murine colitis model, 2-OHOA pretreatment attenuated weight loss, disease activity index, colon shortening, and reduced the spleen index and histological injury scores, whereas OA at the same dose had minimal effects. 2-OHOA also attenuated colonic induction of Il-1β, Il-6, and Tnf-α expression and partially preserved occludin protein levels. Lipidomic analysis showed attenuation of DSS-induced PC depletion, shifts in the PC/PE and LPC/PC ratios, and ceramide and hexosylceramide accumulation. In cytokine-challenged Caco-2 cells, knockdown of the PC biosynthetic enzyme CCTα limited PC preservation by 2-OHOA, while PC(34:1) supplementation attenuated barrier dysfunction, supporting a functional contribution for PC homeostasis to epithelial protection. Together, these findings support preventive effects of 2-OHOA in experimental colitis, accompanied by preservation of lipid homeostasis and epithelial barrier function and reduced inflammatory signaling.
Sterols represent a structurally distinct class of lipophilic natural lipids defined by a rigid tetracyclic scaffold. They are essential for cellular architecture and function, serving as key structural components of biological membranes, precursors for bioactive molecules, and regulators of diverse signaling pathways. However, their high structural similarity and closely related physicochemical properties hamper chromatographic resolution, while the limited availability of reference standards restricts accurate identification and quantification. As a consequence, sterols remain underrepresented in many research areas, including human biomarker studies. To address this, we established a comprehensive sterol analytical toolbox that integrates validated commercially available and in-house synthesized reference standards and is released as an open access sterol library for the research community. The presented library comprises 110 sterols of plant, fungal, and mammalian origin, including six sterol acids, seven epoxy sterols, and 22 oxysterols. Each compound was systematically characterized by chromatographic separation on four different GC-columns, determination of relative retention times and Kováts indices, full scan electron ionization (EI) mass spectra, and optimized multiple reaction monitoring transitions for quantitative analysis. The resulting dataset enabled the establishment of sterol-specific EI-MS fragmentation signatures and retention increment principles, providing a robust framework for the reliable identification of known and previously uncharacterized sterols.
